The present invention relates to a method for DNA synthesis, a kit usable for the method and an article of manufacture, which are useful in the field of genetic engineering, and are capable of shortening a time period needed for polymerase chain reaction (PCR) method.
The synthesis of DNA is used for various purposes in the research of the field of genetic engineering. Among them, much of the DNA syntheses are carried out by the enzymatic method utilizing DNA polymerase, except for chemical synthesis of a short strand DNA such as an oligonucleotide. Accordingly, the DNA polymerase is highly valuable as reagents for DNA sequencing, DNA labelling, and site-directed mutagenesis.
In addition, recently, with the development of PCR method, thermostable DNA polymerases have attracted attention, and various kinds of DNA polymerases suitable for PCR method have been developed and commercialized.
Furthermore, there is known a method capable of carrying out an efficient DNA synthesis by using a combination of plural DNA polymerases, wherein the efficient DNA synthesis could not be achieved by a single DNA polymerase [Proc. Natl. Acad. Sci. USA, 91, 5695-5699 (1994)].
The method is a method using a mixture of DNA polymerases for PCR, the mixture comprising DNA polymerase having 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity (for example, xcex1-type DNA polymerase derived from Pyrrococcus furiosus) and DNA polymerase not having the exonuclease activity (for example, DNA polymerase derived from Thermus aquaticus (Taq DNA polymerase), and is known as LA-PCR method.
According to this method, the yield of amplified DNA is increased, as compared with that by conventional PCR using only one kind of DNA polymerase. The method can also amplify long DNA fragment, which could not be amplified by conventional PCR.
Optimum PCR conditions which have been conventionally and generally performed are shown in Table A. The amplification of each DNA is terminated at a reaction time of about 90 minutes in the 1 kbp amplification, at a reaction time of about 268 minutes in the 10 kbp amplification, and at a reaction time of about 478 minutes in the 20 kbp amplification.
Since the PCR method has an ability to amplify a trace amount of DNA into several millions times the amount in a short time period, the PCR method is applied to all sorts of studies, tests and clinical fields including medical science and agriculture. Particularly, the PCR method is powerful in genetic diagnosis of infectious diseases such as cancers and AIDS, and the like. In addition, its application has been extended even to city life, including confirmation of a guilty party, or a parent-and-child relationship by genetic diagnosis; a gene detection of harmful bacteria in foods, and the like.
However, it has become an important problem to further shorten a reaction time period of PCR and to speed up PCR in food examination for which a quick result is demanded, and in a clinical test in which PCR is required to be carried out on a large scale.
Further, PCR procedures are indispensable for DNA chip preparation, genome analysis and the like, so that it is important to improve its efficiency in view of efficiently carrying out a whole research.
An object of the present invention is to provide a DNA synthesis method, a kit for use in the synthesis method and an article of manufacture, for carrying out rapid PCR with a more shortened time period as compared with conventional PCR.
A first invention of the present invention relates to a DNA synthesis method with a shortened time period required for DNA synthesis by polymerase chain reaction (PCR), characterized in that DNA polymerase is used in an amount effective for providing more than 10 ng of amplified DNA fragments of about 2 kb per 50 xcexcl of a reaction mixture, when PCR is carried out under the following conditions (A) and (B):
(A) reaction mixture: 50 xcexcl volume of a reaction mixture comprising DNA polymerase, 1 ng of genomic DNA from Escherichia coli, and 10 pmol each of primers Eco-1 and Eco-2 (nucleotide sequences of the primers Eco-1 and Eco-2 being shown in SEQ ID NOs: 10 and 11 of Sequence Listing, respectively); and having a composition suitable for the DNA polymerases; and
(B) reaction conditions: 35 cycles of PCR, wherein one cycle consists of 99xc2x0 C., 1 second -66xc2x0 C., 7 seconds.
A second invention of the present invention relates a kit for DNA synthesis usable for the DNA synthesis method of the first invention of the present invention, characterized in that a PCR reagent mixture which is prepared in accordance with instructions of the kit, comprises a DNA polymerase in an amount effective for providing more than 10 ng of amplified DNA fragments of about 2 kb per 50 xcexcl of a reaction mixture, when PCR is carried out under the following conditions (A) and (B):
(A) reaction mixture: 50 xcexcl volume of a reaction mixture comprising DNA polymerase, 1 ng of genomic DNA from Escherichia coli, and 10 pmol each of primers Eco-1 and Eco-2 (nucleotide sequences of the primers Eco-1 and Eco-2 being shown in SEQ ID NOs: 10 and 11 of Sequence Listing, respectively); and having a composition suitable for the DNA polymerase; and
(B) reaction conditions: 35 cycles of PCR, wherein one cycle consists of 99xc2x0 C., 1 second-66xc2x0 C., 7 seconds.
A third invention of the present invention relates to an article of manufacture of a PCR agent, comprising packaging material and a PCR reagent contained within the packaging material, wherein the PCR agent comprises DNA polymerases, and wherein a label or instruction indicates that the PCR reagent can be used for PCR in a short time period, the label being attached to the packaging material, and the instruction being enclosed with the packaging material.
(1) DNA Synthesis Method of the Present Invention
The DNA synthesis method of the present invention is a DNA synthesis method with a shortened time period for synthesizing DNA by PCR method, namely rapid PCR, characterized in that DNA polymerase is used in an amount effective for providing more than 10 ng of amplified DNA fragments of about 2 kb per 50 xcexcl of reaction mixture, when PCR is carried out under the following conditions (A) and (B):
(A) reaction mixture: 50 xcexcl volume of a reaction mixture comprising DNA polymerase, 1 ng of genomic DNA from Escherichia coli, and 10 pmol each of primers Eco- 1 and Eco-2 (nucleotide sequences of the primers. Eco-1 and Eco-2 being shown in SEQ ID NOs: 10 and 11 of Sequence Listing, respectively); and having a composition suitable for the DNA polymerase; and
(B) reaction conditions: 35 cycles of PCR, wherein one cycle consists of 99xc2x0 C., 1 second-66xc2x0 C., 7 seconds.
According to the DNA synthesis method of the present invention, since xe2x80x9ceffective amount of DNA polymerasexe2x80x9d is used, there can be shortened a time period required in synthesis reaction of complementary strand corresponding to a given size of DNA fragment (extension step), i.e. there can be shortened a time period required in 1 cycle of reaction. As a result, there are exhibited excellent effects that in PCR, a desired DNA fragment can be amplified in a short time period which has not been achieved conventionally, in other words, rapid PCR can be carried out with a shortened total time period required in PCR.
In the present invention, the term xe2x80x9ceffective amount of DNA polymerasexe2x80x9d means an amount of a DNA polymerase which is an amount corresponding to a sufficient activity such that an amount of a DNA fragment of about 2 kb is more than 10 ng per 50 xcexcl volume of a reaction mixture, when a 35 cycle PCR is carried out by using 50 xcexcl of a reaction mixture containing 1 ng of E. coli genomic DNA, and 10 pmol each of primers Eco-1 and Eco-2 (nucleotide sequences of primers Eco-1 and Eco-2 being shown in SEQ ID NOs: 10 and 11 of Sequence Listing, respectively), wherein one cycle consists of 99xc2x0 C., 1 second-66xc2x0 C., 7 seconds. Therefore, as long as an amount of DNA polymerase is an amount capable of carrying out PCR under these conditions, an amount of its protein is not limited. The use of this effective amount of DNA polymerase can speed up the PCR.
The term xe2x80x9cE. coli genomic DNAxe2x80x9d described in the present specification includes, for example, genomic DNA prepared from E. coli strain JM109 (Escherichia coli JM109) according to the method described in xe2x80x9cIDENSHIxe2x80xa2TANPAKUSHITSU JIKKENSOUSA BLOTTING (Genexe2x80xa2Protein Experimental Manipulation and Blotting)xe2x80x9d coauthored by Yoshiyuki Kuchino, Hisamaru Hirai, and Ikunosuke Sakuragi, 1987, published by Soft Science Company. In addition, the details of the preparation method are described in Bio View, 13, 6-5 (1994, published by Takara Shuzo Co., Ltd.). Furthermore, the genomic DNA is available as genome DNA set for LA PCR(trademark) from Takara Shuzo Co., Ltd.
Incidentally, as a composition of a reaction mixture, there may be used a reaction mixture having a composition suitable for DNA polymerase used. Here, the term xe2x80x9ccomposition suitable for DNA polymerasexe2x80x9d means a composition capable of providing optimum conditions such as optimum kinds of buffers, optimum pH, optimum salt concentration (magnesium salt, and the like), optimum dNTPs concentration, optimum amount of primers and other additives.
Here, an enzyme activity unit of DNA polymerase is expressed as an index an ability of catalyzing the incorporation of nucleotides (for example, labeled dNTPs) into template DNA. Such a method for determining an activity is described in, for example, DNA Polymerase from Escherichia coli, authored by Richardson C. C., page 264, Procedures in Nucleic Acids Research, 1966, published by D. R. Harper and Row Co., Ltd., edited by Cantoni G. L. et al. For example, as to conditions when an activity of Taq DNA polymerase, which is DNA polymerase derived from Thermus aquaticus is determined by the method, the following conditions are exemplified.
(a) template DNA: activated salmon sperm DNA;
(b) a composition of a reaction mixture: 25 mM TAPS (pH 9.3 at 25xc2x0 C.), 50 mM potassium chloride, 2 mM magnesium chloride, 1 mM xcex2-mercaptoethanol, 200 xcexcM each of dATP, dGTP and dTTP, and 100 xcexcM [xcex1-32p] dCTP, a total volume of 50 xcexcl;
(c) a method for determining an activity: To a reaction mixture having the composition of (b) containing the template DNA of (a) is added a sample containing Taq DNA polymerase, and the resulting mixture is incubated at 74xc2x0 C. for 10 minutes. Thereafter, acid-insoluble substances are collected, and the radioactivity contained in the acid-insoluble substances are determined.
Activated salmon sperm DNA used in the above method for determining an activity is prepared as follows.
{circle around (1)} Salmon testes DNA (manufactured by Sigma Co., Ltd.) is allowed to swell with sterilized water.
{circle around (2)} Seventy units per microliter of DNase I (manufactured by Takara Shuzo Co. Ltd.) is diluted with 150 mM sodium chloride so as to be diluted within a range of 500 times-4000 times, preferably within a range of 1500 times-3000 times.
{circle around (3)} To 20 mg of the above swelled DNA are added 50 mM Tris-hydrochloric acid buffer (pH 7.5), 5 mM magnesium chloride, 0.05% BSA, 50 xcexcl of the above diluted DNase I, so as to make up a volume of 10 ml of a reaction mixture.
{circle around (4)} After treatment at 37xc2x0 C. for 10 minutes, enzyme is inactivated by treating at 77xc2x0 C. for 10 minutes. To a partial portion thereof, perchloric acid (HClO4) is added, so as to have a final concentration of about 0.4 M, and the resulting mixture is centrifuged, thereby providing the resulting supernatant as an A260 sample. The remaining portion is purified by subjecting it to phenol extraction, chloroform/isoamyl alcohol extraction, and ethanol precipitation.
{circle around (5)} Absorbance of UV260 of DNA before the above enzyme treatment (A260 control) and that of the A260 sample obtained in the above item {circle around (4)} are determined to calculate decomposition rate using (A260 sample) /(A260 control)xc3x97100 (%). In this case, as a substrate for determining the above incorporating activity, there can be used an enzyme-treated DNA having a decomposition rate of preferably from about 3% to about 9%, more preferably about 4.5% to about 7.5%.
Incidentally, depending on the kinds of enzymes to be tested for incorporating activity, it is reasonable to select an enzyme-treated DNA having the most optimal decomposition rate in a preliminary experiment using an enzyme-treated DNA having each decomposition rate.
Usually, one unit (hereinafter described as xe2x80x9c1 Uxe2x80x9d) of DNA polymerase activity determined by the above method, namely, an activity for incorporating dNTPs into DNA (hereinafter, simply described as dNTPs-incorporating activity) is defined as xe2x80x9can amount of enzyme capable of incorporating 10 nmol of dNTPs per 30 minutes into acid-insoluble substancesxe2x80x9d.
Conventionally, in PCR using a DNA polymerase, it is standard to add a DNA polymerase in an amount of 1.25 U to 2.5 U as a dNTPs-incorporating activity in 50 xcexcl of a reaction mixture. Although not particularly limited in the present invention, DNA amplification with a short time period conventionally not achieved, namely, speedup of PCR, can be achieved by carrying out PCR with addition of DNA polymerase in an amount of 4 to 20 U as a dNTPs-incorporating activity per 50 xcexcl of a reaction mixture.
An example of rapid PCR of the present invention is shown in Table B. By comparison of Table A with Table B mentioned above, an effect of shortening a time period by the rapid PCR of the present invention is obvious. According to the present invention, there is provided rapid PCR with a shortened total time period for PCR.
In Table B, as polymerase A, there is used DNA polymerase in an amount of 5 U as a dNTPs-incorporating activity per 50 xcexcl (PCR reagent mixture). In addition, in Table B, E. coli genomic DNA is the same as that in Table A.
When the effective amount of DNA polymerase used in the rapid PCR of the present invention is used in amplification of a DNA fragment under conditions for the rapid PCR of the present invention, there is provided an amount of amplified products of the same level as that of carrying out PCR using TaKaRa Ex Taq in an amount of 1.25 U as a dNTPs-incorporating activity per 50 xcexcl as shown in Table A. Therefore, while the activity unit for an effective amount of DNA polymerase used in the present invention shows a higher activity unit in the dNTPs-incorporating activity than that of the prior arts, an activity unit expressed by PCR performance, namely an activity unit expressed by comparison of amounts of amplified products in PCR processes (PCR effective ratio), is of the same level as that of the prior arts.
Embodiments of the DNA synthesis method of the present invention include a method of using one kind of DNA polymerase and a method of using two or more kinds of DNA polymerases. The above method of using two or more kinds of DNA polymerases includes concretely a method of using DNA polymerase having 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity and the other DNA polymerase substantially having no 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity (Proceedings of the National Academy of the Sciences of the USA, supra); a method of using two or more kinds of DNA polymerases each having 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity; a method of using xcex1-type DNA polymerase and non-xcex1, non-pol I type DNA polymerase; and the like. Here, xe2x80x9cthe other DNA polymerase substantially having no 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activityxe2x80x9d includes naturally-occurring DNA polymerase having no 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity or DNA polymerase in which functional portion involved in expression of 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity is artificially modified to exhibit no activity.
The amount of DNA polymerase used in the present invention (in other words, the above xe2x80x9ceffective amount of DNA polymerasesxe2x80x9d) is an amount exceeding an amount of DNA polymerase standardly used in the indication xe2x80x9cdNTPs-incorporating activityxe2x80x9d as described in the instruction manuals, and the like, which are attached to a commercially available enzyme and kit for standard PCR method. Based on this indication of enzyme activity, an effective amount may be used, wherein the effective amount is an amount effective for exhibiting an effect of shortening a time period required in operation.
Concretely, it is desired that the amount of DNA polymerase used in the present invention, in the indication of dNTPs-incorporating activity, is preferably twice or more, more preferably four times or more, the amounts of enzyme used in conventional PCR, from the viewpoint of fully exhibiting the effect of shortening a time period required in operation, and that the amount is preferably 30 times or less, more preferably 20 times or less, the amounts of enzyme used, from the same viewpoint as above. In addition, when two or more kinds of DNA polymerases are used, there may be used an effective amount of each enzyme, or an effective amount as the amount of enzyme for any one of two or more kinds of DNA polymerases. As a result, as shown in the following examples, according to the DNA synthesis method of the present invention, namely rapid PCR, there can be achieved DNA amplification with a short time period, and confirmation of amplified DNA by general agarose electrophoresis, even under such PCR conditions with a shortened time period that amplification of a desired DNA fragment cannot be confirmed by general agarose electrophoresis or the like, when DNA polymerase is used in an amount in the conventional PCR.
The above xe2x80x9ceffective amount of DNA polymerasexe2x80x9d may be determined, for example, as follows. Concretely, regarding to an arbitrary template DNA, PCR is carried out under standard PCR conditions (standard conditions) such as an amount of enzyme, a thermal profile, and the like, which are standardly used for the amplification of the template DNA. Next, an enzyme amount is determined by adjusting a time period for each reaction step under standard conditions; setting PCR conditions so as to shorten a time period required for a whole amplification reaction; thereafter carrying out PCR, with varying amounts of enzyme, wherein the enzyme amount is an amount obtainable in substantially the same level in PCR performance as an amount of amplified products when a standard amount of the enzyme used, so that the enzyme amount determined or an amount not less than the enzyme amount can be defined as an effective amount of DNA polymerase, which is used for the rapid PCR of the present invention. Here, xe2x80x9camount of amplified productsxe2x80x9d is quantified by, for example, subjecting a given amount of a sample obtained after termination of PCR to electrophoresis; staining the resulting gel after electrophoresis by ethidium bromide to visualize the band ascribed to amplified products; and thereafter measuring fluorescent intensity of the band with the use of instrument, such as an image analysis device (image analyzer) or a densitometer, the instrument being capable of quantifying an amount of a DNA fragment contained in the band which is separated on the gel. Furthermore, an amount of a DNA fragment may be quantified by a known DNA quantification method after purification of amplified products in the sample.
In the rapid PCR of the present invention, the reaction time which is set for each step is not particularly limited, as long as the reaction time is relatively short, as compared to the reaction time under the standard conditions, and exhibits the same level of the enzyme activity as represented by the PCR performance. In the present invention, it is possible to set PCR conditions such that the reaction time for all processes, namely a total time period for PCR is one-half to one-quarter or less of the conventional total time period for PCR. For instance, in the case of amplification of a 1 kbp fragment, while conventional one cycle requires 3 minutes, one cycle in the rapid PCR of the present invention can be shortened to 40 seconds, so that the total time period for PCR is two-ninth of the conventional one. In the case of amplification of a 2 kbp fragment, while conventional one cycle requires about 9 minutes, one cycle in the rapid PCR of the present invention can be shortened to about 1.8 minutes, so that the total time period for PCR is about one-fifth of the conventional one. In the case of amplification of a 10 kbp fragment, while conventional one cycle requires about 16 minutes, one cycle in the rapid PCR of the present invention can be shortened to about 3 minutes, so that the total time period for PCR is about one-fifth of the conventional one.
Conventionally, as enzymes possessing excellent DNA synthesizing speed and capable of amplifying DNA in a short time period, there has been known an enzyme composition comprising DNA polymerase (KOD DNA polymerase) derived from Pyrococcus sp. KOD 1 (Japanese Patent Laid-Open No. Hei 10-42874, trade name: KOD Dash DNA polymerase, manufactured by TOYOBO CO., LTD.). However, no amplified fragments can be visibly confirmed and the amount of the amplified product as quantified by high-sensitive image analyzer is 10 ng or less, even if the enzyme composition were used under the above conditions in a standard amount used of 2.5 U/50 xcexcl as described in the article of manufacture in accordance with the instruction manual, and thereafter the resulting reaction mixture is subjected to general agarose electrophoresis. On the other hand, when the enzyme is used at an xe2x80x9ceffective amount of the DNA polymerasexe2x80x9d as defined in the present invention, even under the above conditions, amplified fragments can be confirmed by agarose electrophoresis, and the amount of its amplified product is an amount exceeding 10 ng.
The DNA polymerase which can be used for the DNA synthesis method of the present invention (namely the rapid PCR) is not particularly limited, and includes, for instance, pol I-type DNA polymerases (E. coli DNA polymerase I, Klenow fragment, Taq DNA polymerase and the like), xcex1-type DNA polymerases [xcex1-type DNA polymerase derived from the above-mentioned Pyrococcus furiosus, DNA polymerase derived from Thermococcus litralis (VENT DNA polymerase), DNA polymerase derived from Pyrococcus sp. KOD1 (KOD DNA polymerase), DNA polymerase derived from Pyrococcus sp. GB-D (DEEP VENT DNA polymerase), and the like], and non-xcex1, non-pol I type DNA polymerases not belonging to any of these polymerases. Incidentally, each of the pol I type DNA polymerases and xcex1-type DNA polymerases refers to a group of enzymes classified by the homology on the amino acid sequences thereof, and the feature on the amino acid sequences is described in Nucleic Acids Research 15, 4045-4057 (1991).
In addition, the non-xcex1 non-pol I type DNA polymerase includes, for instance, DNA polymerase derived from Pyrococcus furiosus disclosed in WO97/24444 Pamphlet.
The DNA polymerase which can be used for the method of the present invention is not limited to a single kind of a DNA polymerase, and two or more kinds of DNA polymerases can be also used as a DNA polymerase composition by, for instance, mixing a DNA polymerase possessing 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity with another DNA polymerase possessing substantially no 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity. A mixing ratio of both the enzymes is not particularly limited, and it may be a mixing ratio appropriate for the rapid PCR of the present invention depending upon the kinds of both the enzymes. It may be used in a ratio of the DNA polymerase possessing 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity to the other DNA polymerase possessing substantially possessing no 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity in the range of 9:1-1:500. As an example of a polymerase composition thereof, there can be suitably used TaKaRa Ex Taq DNA polymerase (manufactured by Takara Shuzo Co., Ltd.).
Generally in PCR, DNA amplification is carried out by three-step reaction of dissociation (denaturation) of a double stranded template DNA to a single stranded one, annealing of a primer to a single stranded template DNA, and a complementary strand synthesis (extension) from the primer. In addition, DNA amplification is also carried out in a so-called xe2x80x9cshuttle PCRxe2x80x9d [xe2x80x9cPCR Hou Saizensen (PCR Method Frontier),xe2x80x9d xe2x80x9cProteins, Nucleic Acids, Enzymesxe2x80x9d an extra number 41, No. 5, 425-428 (1996)], which is a two-step reaction in which the annealing step of the primer and the extension reaction step among the three-step reactions described above are carried out at the same temperature. In the DNA synthesis method of the present invention, since the time period required for the above extension step can be particularly shortened, in any of the above-mentioned three-step reaction and two-step reaction, the time period required for an entire synthesis reaction can be shortened.
In the DNA synthesis method of the present invention, when the DNA synthesis reaction is carried out, the DNA synthesis can be further efficiently carried out by subjecting to the DNA synthesis reaction in the presence of a substance possessing the action of enhancing the DNA-synthesizing activity owned by the DNA polymerase, namely a substance for achieving higher performance in the rapid PCR.
As one substance for achieving higher performance in the rapid PCR, there is included a substance having electrically negative charges or a salt thereof, particularly an acidic substance or a salt thereof.
PCR is carried out in the presence of an effective amount of the acidic substance and/or a salt thereof, whereby the conditions for speedup can be universalized. In other words, high-performance rapid PCR can be carried out without being affected by the nature of the template (for instance, GC content, or the like). In addition, at least one kind selected from spergualins, degradation products and salts thereof may be selected.
When the rapid PCR of the present invention is carried out, the details on the function of an effective amount of an acidic substance and/or a salt thereof, for instance, an acidic substance having a sugar backbone structure, are unknown. When an effective amount of the DNA polymerase for carrying out the rapid PCR of the present invention is used, since excess DNA polymerase is trapped with the acidic substance during the DNA synthesis reaction, the high-performance rapid PCR can be further achieved by supplying the most appropriate DNA polymerase for PCR to a template DNA by the effects of the acidic substance.
The action of the acidic substance or a salt thereof of enhancing DNA-synthesizing activity can be evaluated by the size of the DNA strand newly synthesized per unit time period or the amount of the amplified products in PCR. In the DNA synthesis method of the present invention, the above-mentioned acidic substance or a salt thereof is used in an amount effective for exhibiting its action. The effective amount can be evaluated by comparing, for instance, the amount of the amplified products in the case where PCR is carried out by using a reaction mixture in which various amounts of the above acidic substance or a salt thereof are added, with the amount of the amplified products in the case where PCR is carried out without adding these substances. The amount of the amplified products can be quantified, for instance, by subjecting a given amount of the reaction mixture after PCR to electrophoresis, staining the gel after electrophoresis by ethidium bromide or the like, and determining the intensity of fluorescence of the band ascribed to the amplified products by using an imaging analyzer or the like.
The acidic substance possessing an action of enhancing DNA-synthesizing activity is not particularly limited. For instance, acidic macromolecular substances such as acidic polysaccharides can be used. In addition, polyglutamates, polyacrylates, polyvinyl sulfates, polystyrene sulfates, and DNAs not serving as a template for a desired DNA synthesis can be also used. Incidentally, in the present specification, the acidic substance also encompasses a salt of the above acidic substances, as long as it possesses an action for enhancing DNA-synthesizing activity. The acidic polysaccharides which can be used in the present invention include, for instance, sulfate group-containing sulfated polysaccharides representatively exemplified by sulfated-fucose-containing polysaccharides, dextran sulfate, carrageenan, heparin, heparan sulfate, rhamnam sulfate, chondroitin sulfate, and dermatan sulfate (chondroitin sulfate B); polyuronic acids such as hyaluronic acid, alginic acid and pectin, and the like. As the above sulfated-fucose-containing polysaccharides, there can be used sulfated-fucose-containing polysaccharide-F or sulfated-fucose-containing polysaccharide-U. Here, the term xe2x80x9csulfated-fucose-containing polysaccharide-Fxe2x80x9d refers to a sulfated-fucose-containing polysaccharide substantially containing no uronic acid, obtainable from the plant of Phaeophyceae, for instance, by the method disclosed in WO97/26896 Pamphlet, or by the method disclosed in WO97/47208 Pamphlet. In addition, the term xe2x80x9csulfated-fucose-containing polysaccharide-Uxe2x80x9d refers to a sulfated-fucose-containing polysaccharide containing uronic acid, obtainable by the method described in the above Pamphlets.
The salt of the above acidic substance is not particularly limited, as long as it possesses an action of enhancing DNA-synthesizing activity, and a water-soluble salt is preferable. The water-soluble salt includes, for instance, alkali metal salts such as sodium dextran sulfate, sodium alginate, sodium polyglutaminate, sodium heparin, potassium dextran sulfate and lithium heparin.
The above acidic substance may be, for instance, naturally occurring products, or chemically or enzymatically synthesized products, as long as the acidic substance is a substance keeping the action of enhancing the DNA-synthesizing activity. The above acidic substance may be any of unpurified products containing the same, partially purified products or purified products. Further, the acidic substance may be subjected to an appropriate modification in a range in which an action of enhancing DNA-synthesizing activity is kept. In addition, the acidic substance used in the present invention may be a substance obtained by subjecting it to degradation procedures so that the molecular weight of the above-mentioned acidic substance is in an appropriate form for exhibiting an action of enhancing DNA-syntbesizing activity, or the acidic substance may be a substance obtained by further subjecting the product after the degradation procedures to a fractionation by molecular weight, as long as the acidic substance possesses an action of enhancing DNA-synthesizing activity. In the present invention, an acidic substance having a molecular weight of several thousands or more can be preferably used. Further, these substances can be used alone or in admixture.
The above acidic substance possessing an action of enhancing DNA-synthesizing activity is added for the purpose of efficiently exhibiting an activity of the DNA polymerase, or of keeping its activity in the rapid PCR of the present invention. The amount added can be optimized depending upon the kinds of the acid substances, and the acidic substance may be added at 0.1 ng to 100 xcexcg, preferably at 1 ng to 10 xcexcg, per 50 xcexcl of the reaction mixture. The action of the acidic substance is not particularly limited, and it is considered to be on the bases of holding the DNA polymerase on its molecule, thereby suppressing the nonspecific interaction of the DNA polymerase to a template DNA, and of providing an optimal amount of the DNA polymerase for the template DNA. In other words, the DNA synthesis reaction efficiently progresses by optimizing the interaction between the template DNA and the DNA polymerase, the interaction increasing with the progress of the DNA synthesis reaction.
Further, there are exhibited effects that the influences of amplified regions, nucleotide sequences of the primer, and the like are reduced, thereby stably obtaining amplified products.
The DNA polymerase in which the above acidic substance enhances its activity is not particularly limited. For instance, the above acidic substance can be applied to the DNA synthesis method using various DNA polymerases mentioned above.
In the present invention, there may be added and used to a PCR mixture spergualins and/or salts thereof for the purpose of efficiently exhibiting the activity of the DNA polymerase in the rapid PCR of the present invention, or maintaining its activity.
The spergualins possessing an action of enhancing DNA-synthesizing activity are not particularly limited. Examples thereof include a 15-deoxyspergualin compound represented by the following general formula (I):
Guxe2x80x94(CH2)6xe2x80x94CONHCH(OR)CONH(CH2)4NHxe2x80x94(CH2)3xe2x80x94NH2xe2x80x83xe2x80x83(I)
wherein Gu is guanidino group, and R is hydrogen atom or methyl group, or a salt thereof, and the like.
As the above spergualins, for instance, 15-deoxyspergualin, where R of the above general formula (I) is hydrogen atom, or a salt thereof, is preferable.
In addition, the salt of the spergualins may be a salt with an inorganic acid or a salt with an organic acid, as long as the salt is a substance capable of exhibiting an action of enhancing DNA polymerase activity.
Incidentally, the above spergualins are derivatives of spergualin isolated from producing bacteria of the genus Bacillus, which is a substance known to possess anti-tumor activity, immunoenhancement activity, and immunosuppression activity depending upon the kinds of the derivatives (Japanese Patent Laid-Open Nos. Sho 58-62152, Sho 61-129119 and Sho 64-90164). Therefore, these spergualins can be prepared by readily purifying by a known method, or synthesizing by a known method.
A process for preparing the above 15-deoxyspergualin or a salt thereof is disclosed, for instance, in Japanese Examined Patent Publication No. Sho 61-23183 or Example 6 of U.S. Pat. No. 4,603,015, and the like.
The above spergualins may be naturally occurring products or chemically or enzymatically synthesized products, as long as spergualins are a substance keeping an action of enhancing DNA-synthesizing activity. The above spergualins may be any of unpurified products containing the same, partially purified products or purified products. Further, the above spergualins may be subjected to appropriate modifications or a degraded product in a range in which an action of enhancing DNA-synthesizing activity is kept. Further, these substances can be used alone or in admixture.
In the present specification, the degradation product of the spergualins Is not particularly limited, as long as the degradation product is a substance keeping an action of enhancing DNA-synthesizing activity. The degradation product includes, for instance, a substance formed by hydrolyzing the spergualins at room temperature under strongly alkali condition using sodium hydroxide, a substance formed by hydrolyzing the spergualins with heating under weakly alkali conditions such as buffers for PCR, and the like. The substance formed by hydrolysis under each of the above conditions is not particularly limited. When the above 15-deoxyspergualin compound represented by the general formula (I) is used, the substance includes, for instance, a compound represented by the general formula (II):
Guxe2x80x94(CH2)6xe2x80x94CONH2xe2x80x83xe2x80x83(II)
wherein Gu is the same group as that of the general formula (I);
a compound represented by the general formula (III):
HOxe2x80x94CH(OR)CONH(CH2)4NHxe2x80x94(CH2)3xe2x80x94NH2xe2x80x83xe2x80x83(III)
wherein R is the same group as that of the general formula (I);
a compound represented by the general formula (IV):
OHCxe2x80x94CONH(CH2)4NHxe2x80x94(CH2)3xe2x80x94NH2xe2x80x83xe2x80x83(IV)
and the like. The degradation product of the above spergualins also encompasses a salt of the degradation product of the spergualins, as long as it possesses an action of enhancing DNA-synthesizing activity.
The amount of the above spergualins used is not particularly limited, as long as it is in a range capable of exhibiting an action of enhancing DNA-synthesizing activity. The amount used may be an amount so as to have an optimal concentration depending upon the kinds and amounts of the template DNA used, the length of a region to be amplified, and the kinds of the DNA polymerases. For instance, in the case of 15-deoxyspergualin trihydrochloride, it may be added so as to have a final concentration of 0.1 xcexcM to 500 xcexcM, preferably 20 xcexcM to 100 xcexcM.
The action of the spergualins of the present invention is not particularly limited, and it is thought to efficiently exhibit the activity of the DNA polymerase or to hold the DNA polymerase, thereby suppressing nonspecific interaction of the enzyme to the DNA. Also, it is thought to act to the complex of template DNA and the primer, thereby facilitating the primer extension reaction.
When the above spergualins and the acidic substance are used in combination, both parties may react to form a salt, and it may be a substance possessing an action of enhancing DNA-synthesizing activity.
When the above spergualins and the acidic substance are used in combination, although there are no particular limitations in the amount, as long as the amount is in a range capable of exhibiting an action of enhancing DNA-synthesizing activity, it may be an amount so as to have optimal coexisting ratio depending upon the kinds and amounts of the template DNA used, the length of a region to be amplified, the kinds of the DNA polymerases, and the like.
Incidentally, the above spergualins or a salt thereof, and a mixture comprising the above substance having electrically negative charges or a salt thereof and the above spergualins or a salt thereof can be used as DNA-synthesizing activity enhancers.
Regarding the DNA synthesis method described above, the method of the present invention also encompasses the detail description of the method, for instance, a method for preparing a PCR reagent mixture, a provision of printed matters describing information such as recommended reaction conditions and an act of instructing the method of the present invention through electronic media such as internet.
(2) Kit Usable for DNA Synthesis Method of Present Invention
Rapid PCR can be carried out by using the kit of the present invention. The kit as mentioned above is not particularly limited, as long as it is a kit usable in the reaction with the DNA synthesis by PCR method, and includes a kit for rapid PCR for carrying out in vitro DNA synthesis reaction.
The kit of the present invention is a kit usable in in vitro DNA synthesis, wherein the PCR reagent mixture prepared in accordance with the instruction of the kit comprises xe2x80x9can effective amount of the DNA polymerasexe2x80x9d used in the DNA synthesis method described in item (1) above, i.e. an effective amount of a DNA polymerase such that an amount of a DNA fragment of about 2 kb is more than 10 ng per 50 xcexdl of a reaction mixture, when 35 cycles of PCR is carried out by using 50 xcexcl volume of a reaction mixture containing 1 ng of E. coli genomic DNA, and 10 pmol each of primers Eco-1 and Eco-2, wherein one cycle consists of 99xc2x0 C., 1 second-66xc2x0 C., 7 seconds.
The PCR reagent mixture prepared by using the kit of the present invention is not particularly limited, and for instance, the reagent mixture comprises 4 to 10 U as dNTPs-incorporating activity of a DNA polymerase for rapid PCR in 50 xcexcl of the reaction mixture, including, for instance, TaKaRa EX Taq DNA polymerase. In addition, as the composition for the reaction mixture, a reagent mixture having a composition appropriate for the DNA polymerase used may be used.
The above xe2x80x9cinstructionxe2x80x9d refers to a printed matter describing a method of use of the kit, for instance, a method for preparing a PCR reagent mixture, recommended reaction conditions, and the like, which may take a form, beside""s pamphlet or leaflet type of instruction manuals, of a label attached to the kit, a packaging material enclosing the kit, and the like. Further, it also encompasses information disclosed or provided through electronic media such as internet. Regarding the preparation of the PCR reagent mixture, the kit of the present invention encompasses a kit to which instructions for the use of the amount of the above DNA polymerase and/or the addition of the above acidic substance or a salt thereof are attached, or a kit for which the method of the present invention is disclosed and provided through electronic media such as internet.
In addition, the kit may contain an acidic substance or a salt thereof possessing an action of enhancing DNA-synthesizing activity of the DNA polymerase. As the acidic substance or a salt thereof, ones described in item (1) above can be used. The acidic substance or a salt thereof as mentioned above efficiently allows to exhibit the DNA polymerase activity or to hold the enzyme, whereby the interaction between the DNA and the enzyme can be properly regulated. Therefore, the performance of the kit of the present invention is further improved.
In addition, the kit may comprise at least one kind selected from the group consisting of spergualins, degradation products thereof, and salts thereof.
When the above spergualins and the acidic substance are used in combination, the both parties may react to form a salt, and they may be in any forms as long as they are substances possessing an action of enhancing DNA-synthesizing activity.
The DNA polymerase included in the present invention is not particularly limited, and includes various kinds of DNA polymerases for rapid PCR shown in item (I) above.
The kit may comprise a reagent necessary for the reaction of the DNA polymerase such as dNTPs, magnesium chloride, and buffer components for keeping the reaction mixture at an appropriate pH. The above DNA polymerase, the acidic substance and other reagents may be contained in the kit in a state where each is present as an independent component, or a state in which some of the components are combined, including, for instance, a state in which the components are added to the reaction buffer and the like.
One embodiment of the kit of the present invention includes a composition comprising, besides the above DNA polymerase, various components necessary for DNA synthesis by PCR method, including, for instance, dNTP, magnesium chloride, buffer components for keeping the reaction mixture at an appropriate pH, and the like. The composition may further comprise the above acidic substance. The composition described above can be prepared by adding a primer for amplifying a desired DNA fragment and template DNA, and further adding, as occasion demands, water or buffer, whereby a reaction mixture can be prepared. Furthermore, when the DNA fragment to be amplified by the kit is determined, the composition may comprise a primer appropriate for the amplification of the fragment. By the use of the composition described above, the DNA synthesis reaction, namely the rapid PCR, can be extremely conveniently and rapidly carried out.
When applied to manipulations such as PCR and sequencing utilizing PCR, DNA labeling, cDNA synthesis, and site-directed mutagenesis, the kit of the present invention exhibits an excellent effect that the time period required for the manipulations can be shortened. For instance, when the above kit is applied to PCR, the time period required for amplification of DNA of the same chain length is shorter than that by conventional PCR method or LA-PCR method. Therefore, even under rapid PCR conditions where the amplification of DNA was impossible in the conventional method, the amplification of DNA can be carried out. In addition, since the kit of the present invention can shorten the time period required for the entire amplification reaction, there is exhibited an excellent effect that gene diagnostic method or the like can be carried out at a shorter time period by the use of the gene diagnostic method on the basis of PCR method.
(3) Article of Manufacture of Rapid PCR Agent of the Present Invention
The article of manufacture of a PCR agent of the present invention is an article of manufacture of a PCR agent, comprising packaging material and a PCR reagent contained within the packaging material, wherein the PCR agent comprises DNA polymerases, and wherein a label or instruction indicates that the PCR reagent can be used for PCR in a short time period, the label being attached to the packaging material, and the instruction being enclosed with the packaging material. The above PCR agent may comprise a DNA polymerase and buffer appropriate for the DNA polymerase and/or dNTP. Therefore, one of ordinary skill in the art can conveniently carry out the rapid PCR of the present invention by following the label indicated on the article of manufacture or the instruction manual attached to the manufacture, and the article of manufacture s useful in various industrial fields requiring the rapid PCR of the present invention.
According to the DNA synthesis method (rapid PCR) of the present invention, the time period required for the entire amplification reaction can be shortened. Although it depends upon the performance of the apparatus used, for instance, a total time period for PCR required for amplifying a DNA of 2 kb is shortened to about one-half that of conventional method, and a total time period for amplifying a DNA of about 20 kb is shortened to about one-fifth that of conventional method, whereby speed-up of PCR can be achieved for the first time. The method of the present invention exhibits an excellent effect that gene diagnostic method can be carried out in a shorter period of time by, for instance, the use of gene diagnostic method on the basis of PCR method. The method is particularly suitable for nested PCR or the like in which PCR is carried out twice.
The rapid PCR of the present invention is extremely useful in the development and manufacture of the techniques requiring a large amount of PCR manipulations such as a DNA chip. The DNA chip comprises a glass chip of a size of thumb and about 10000 kinds of DNAs, wherein the DNAs are immobilized on the glass chip. In order to manufacture the DNA chip, it is necessary that DNAs requiring spots are amplified and prepared by amplification with PCR, and the necessary PCR manipulations therefor would be enormous. For instance, if 10000 of DNA chips described above are supposedly made, there is exhibited an effect that the chips can be manufactured in a short time period of about 3 weeks according to the rapid PCR of the present invention, in contrast to an overall period of about 3 months just for PCR manipulations when the conventional PCR is employed.
In addition, the PCR manipulations required for elucidating an entire sequence of genome of Bacillus subtilis (5000000 bases) can be achieved in about 3 weeks according to the rapid PCR of the present invention, in contrast to about 3.5 months in the conventional PCR, thereby showing a great difference.
In the rapid PCR of the present invention, PCR can be carried out by using conventional PCR apparatus without using specialized apparatus. The rapid PCR of the present invention is excellent in the rapidness and the reactivity, and is an extremely useful technique as high-sensitivity PCR.
The present invention will be hereinbelow described in further detail by means of the working examples, without intending to restrict the scope of the present invention to these working examples.
In the following working examples, the activities of the commercially available DNA polymerases were represented on the basis of the indicated units in xe2x80x9cdNTPs-incorporating activityxe2x80x9d described in instruction manual for each enzyme product. In addition, unless specified otherwise, the reaction solution comprising a commercially available enzyme was prepared in accordance with the manual for each enzyme, or prepared by using a reaction buffer attached thereto. PCR was carried out by using TaKaRa PCR Thermal Cycler PERSONAL (manufactured by Takara Shuzo Co., Ltd.) unless specified otherwise.